We have discovered the first-ever inhibitors of PF4, a platelet protein central to the pathophysiology of heparin induced thrombocytopenia (HIT). Heparin is a naturally-occurring anticoagulant that prevents the formation of clots and extension of existing clots within the vasculature, and major medical applications of heparin include dialysis, cardiac catheterization, and cardiopulmonary bypass surgery. Heparin therapy is usually safe and effective; however some patients develop HIT as a serious complication caused by an immunological reaction that targets platelets leading to a low platelet count (thrombocytopenia). HIT increases the risk of blood clots forming within blood vessels and blocking the flow of blood (thrombosis), referred to as HITT when thrombosis occurs. HITT develops in approximately 1-3% of patients treated with heparin for 5-10 days. Affected individuals have a 20-50% risk of developing new thromboembolic events, a mortality rate ~20%, and an additional ~10% of patients require amputations or suffer other major morbidity. Current treatment for HIT relies on elimination of heparin exposure from patients with suspected HIT and administration of direct thrombin inhibitors, which carry a significant risk of bleeding. Despite the removal of heparin from these patients, they remain at significant risk for thrombosis and death. We have discovered a novel approach to the treatment of HIT via inhibition of PF4 by destabilizing the functionally-active PF4 tetramers, forming the inactive monomers and dimers. The tetrameric form of PF4 binds to heparin to form Ultralarge Complexes (ULC). We have recently reported on the identification and characterization of PF4 antagonists (PF4As) which disrupt PF4 tetramerization, which is a prerequisite for ULC formation, and inhibit the formation of ULC. This grant proposal is to validate PF4 as a target for HIT by designing and preparing new analogs of the PF4 antagonist PF4A01 that have improved potency and similar or better ADME and drug-like properties as novel therapeutics for the treatment and/or prevention of this devastating complication of heparin therapy. Using the combined expertise of Fox Chase Chemical Diversity Center, Inc. in medicinal chemistry and drug discovery, and the Sachais Laboratory at the University of Pennsylvania in HIT-based approaches, we anticipate this Phase I project to result 2 - 3 improved PF4 lead antagonists to form the basis of a full drug discovery program in Phase II. The first aim is to identify structure activity relationships (SARs) for the inhibition of PF4 tetramerization and ULC formation in order to increase potency in the in vitro assays (IC50 1 M). Aim 2 is to test analogues in in vitro assays for targeted activities (inhibition of PF4 tetramerization, ULC inhibition, and inhibition of cellular activation), and Aim 3 is to characterize and improve acceptable ADME drug values for: metabolic stability (microsomes), aqueous solubility, lipophilicity (cLogP), plasma protein binding, CypP450 inhibition (3A4, 2C9, 2D6), and in vivo pharmacokinetics in mice after i.v. administration. After Phase II, we would seek to enter pre-clinical development prior to filing an IND application with the FDA.